JP2002524072A - Human prothrombin-derived endothelial cell growth inhibitor - Google Patents

Abstract

  (57) [Summary] Human prothrombin kringles 1 and 2 having anti-endothelial cell proliferation and antitumor activity, cDNA encoding human prothrombin kringles 1 and 2 derived from human prothrombin, a recombinant expression vector containing this cDNA, and human prothrombin kringle A recombinant microorganism transformed with a recombinant expression vector containing cDNA encoding -1 or 2, human prothrombin kringles -1 and 2 from human plasma, and recombinant human prothrombin kringle-from recombinant microorganism And a pharmaceutical use of the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to human prothrombin kringles -1 and 2 having anti-endothelial cell proliferative and antitumor activity, and more specifically, cDNA encoding human prothrombin kringles -1 and 2 derived from human prothrombin, A recombinant expression vector containing the same, a recombinant microorganism transformed with a recombinant expression vector containing the gene encoding human prothrombin kringle-1 or 2, and human prothrombin kringle-1 or 2 prepared from human plasma. The present invention relates to a method for producing recombinant human prothrombin kringle-2 from a recombinant microorganism, and a pharmaceutical application of the protein.

[0002] Blood coagulation is a reaction to vascular injury that causes the activation of platelets and coagulation factors by the final formation of fibrin plugs. Some clotting factors are vitamin K for normal biosynthesis
Is a zymogen of a serine protease. The active forms of these proteases and their cofactors form a membrane-bound macromolecular complex. In the final step, prothrombin is activated to thrombin by a protease. Subsequently, thrombin cleaves the designated peptide bond in fibrinogen to form fibrin monomers which are polymerized into insoluble fibrin strands. Coagulation and anticoagulation proteases, Factors VII, IX and X, and protein C contain a gamma-carboxyglutamic acid ("Gla")-containing region at the N-terminus followed by two regions consistent with epidermal growth factor ("EGF"). While having a common region, the C-terminal half of each protein is occupied by a trypsin-like serine protease region.

Prothrombin, a 72 kDa glycoprotein having 581 amino acids, is transformed into thrombin on a charged membrane by the proteolytic activity of prothrombinase and by an enzyme complex comprising protease factor Xa, cofactor calcium, factor Va and the membrane surface. (Mann, KG et al., Ann. Rev. Biochem., 57: 915-956 (1988)
Mann, KG et al., Blood, 76: 1-16 (1990)). Like other coagulation and anticoagulation proteases, prothrombin is organized into several distinct functional regions that are homologous to those found in other plasma serine proteases. The functional region in prothrombin is a Gla region (residues 1-44), two adjacent kringle regions (residues 45-155 and 156-273), and a serine protease catalytic region (residue 274) in the carboxyl-terminal region. Or 581)
Mann, KG, Methods Enzymol., 45: 123-156 (1976)).

[0004] Factor Xa of the prothrombinase complex binds prothrombin to two peptide binding sites, one between Arg273 and Thr274 and the other as Ar
Cleavage between g322 and Ile323 results in an amino-terminal fragment (residues 1-273) designated fragment 1-2 and a carboxy-terminal active thrombin (residues 274-581). Thrombin formed during activation converts fragment 1-2 into fragment 1 (residues 1 to 155) containing the Gla region and kringle-1 region and fragment 2 (residues 156 to 273) containing kringle-2. Further cut (Mann, KG, Methods Enzymol., 45: 123-1
56 (1976); Taneda, H. et al., J. Biochem. (Tokyo), 116: 589-597 (199).
4)).

[0005] Although the methods of transforming prothrombin to thrombin in blood coagulation have been intensively studied, the exact role of prothrombin fragments 1 and 2 is limited to the recognition elements and structural units for macromolecular assembly. Remains indistinct (Hoover (H
oover), GJ et al., Biochemistry, 32: 10936-10943 (1993)).

[0006] Kringles are small protein regions with three disulfide bonds characteristic of the secondary structure found in serine proteases involved in blood coagulation and fibrinolysis (Furie, B. and Furie). ), BC, Cell, 53: 505-
518 (1988)). This kringle region has also been found in other proteins having biological functions different from proteases, like apolipoprotein and hepatocyte growth factor (HGF) (Ikeo, K. et al., J. Am. Mol. Evol.,
40: 331-336 (1995)). Have proposed that these kringle regions are found in individual folding units, but the general functional role for kringles is unknown (DeSerrano, VS et al., Arch. B.
iochem. Biophys., 294: 282-290 (1992)). Plasminogen kringle-1
And four regions, they were known to be involved in the binding of plasminogen to fibrin (Vali, Z. et al., J. Biol. Chem., 259: 13690-13694 (19
84)). These kringle regions are presumed to interact with fibrin by binding to exposed lysine side chains. However, it has been reported that the prothrombin kringle region was not adsorbed on a lysine-sepharose column despite its very similar amino acid sequence (Arni, RK et al., Biochemist.
ry, 32: 4727-4737 (1993)).

[0007] Thus, due to the different nature of the apparently very similar polypeptides, recent work has enabled the application of recombinant DNA technology to express the kringle region and its structure- Trying to understand functional relationships (see Desarrano, VS et al., Arch. Biochem. Biophys., 294: 282-290 (1992))
.

On the other hand, Cao et al. Reported that each region of human plasminogen kringle exhibited an anti-capillary endothelial cell proliferation activity applicable as an anti-cancer agent (Kao, Y.
J. Biol. Chem., 271: 29461-29467 (1996); Boehm, T. et al., Nature.
390: 404-407 (1997)). It has also been demonstrated that the recombinant rabbit prothrombin kringle-2 region has a potent endothelial cell growth inhibitory activity similar to angiostatin (Lee, TH et al., J. Biol. Chem., 273: 288
05-28812 (1998)).

In addition, angiostatin, an angiogenesis inhibitor that inhibits endothelial cell proliferation outside the body and suppresses tumor transfer in the body, is derived from the four first kringle regions (1-4 kringle regions) of plasminogen. (O'Reilly)
, MS et al., Cell, 79: 315-328 (1994)).

[0010] Endothelial cell proliferation is closely related to the angiogenesis process, which is very necessary for tumor growth and maintenance (Folkman, J.
., Nat. Med., 1: 27-31 (1995)). The angiogenesis process is a process of angiogenesis in which new blood vessels grow from existing blood vessels. Capillaries are mainly composed of endothelial cells that are normally quiescent in mammals matured under physiological conditions. The angiogenesis process is required for various physiological processes such as embryo development, wound healing, tissue regeneration, and organ regeneration. Early growth of new blood vessels in pathological conditions can cause tumor growth, diabetic retinopathy, tissue and organ malformation, and cardiovascular disease (Folkman, J. et al., J. Biol.
Chem., 267: 10931-10934 (1992)).

Therefore, extensive research has been conducted on anti-endothelial cell proliferation molecules related to the kringle region. In this regard, anti-angiogenic therapy for tumor endothelial cells
Because they induce little drug resistance, human prothrombin fragments 1 and 2 have been proposed as the most promising candidates for cancer treatment.

The present inventors have isolated human prothrombin kringles-1 and 2 from human plasma, and have shown that the kringle region has an anti-endothelial cell proliferation effect. Subsequently, the inventors determined that the cDs encoding human prothrombin kringle-1 and 2
A recombinant expression vector containing NA, ie, cDNA, was constructed to demonstrate that this recombinant human prothrombin kringle-2 has antitumor activity in vivo. Furthermore, the present inventors synthesized a peptide having the amino acid sequence of human prothrombin kringle-2 and determined an active region for suppressing endothelial cell growth. Therefore, the present inventors have found that the protein or peptide can be applied to the development of an antitumor agent.

Accordingly, a first object of the present invention is to provide human prothrombin kringles-1 and 2 having anti-endothelial cell proliferation and anti-tumor activity.

A second object of the present invention is to provide a cDNA sequence encoding the above human prothrombin kringles-1 and 2.

A third object of the present invention is to provide a recombinant expression vector containing the cDNA.

A fourth object of the present invention is to provide a recombinant microorganism transformed with the recombinant expression vector for expressing human prothrombin kringle-2.

A fifth object of the present invention is to provide a human prothrombin kringle-human prothrombin.
The purpose of the present invention is to provide a peptide derived from No. 2 which suppresses endothelial cell proliferation.

[0018] A sixth object of the present invention is to provide human prothrombin kringles-1 and 2 from human plasma.
And a method for producing recombinant human prothrombin kringle-2 from the recombinant microorganism.

A seventh object of the present invention is to provide an antitumor agent containing an active ingredient of prothrombin kringle-1, 2 or a functionally equivalent peptide thereof.

The above and other objects and features of the present invention will become apparent from the following description given in conjunction with the accompanying drawings. FIG. 1 is a photograph showing a human prothrombin derivative analyzed and purified from human plasma by SDS-PAGE. FIG. 2 is a photograph showing Western blot analysis of human prothrombin and its derivatives. FIG. 3 is a graph showing the endothelial cell growth inhibitory effect of human prothrombin and its derivatives. FIG. 4 shows the anti-angiogenic effect of human prothrombin kringle-1 and 2 on CAM. FIG. 5 is a photograph showing suppression of tumor growth by human prothrombin kringle-2. FIG. 6 is a graph showing suppression of tumor growth by human prothrombin kringle-2.

The present inventors have isolated human prothrombin fragments 1 and 2 containing the kringle region from human plasma and have shown that these fragments have anti-endothelial cell proliferation activity. Hereinafter, the human prothrombin fragments 1 and 2 containing the kringle region are referred to as "human prothrombin kringle-1" and "human prothrombin kringle-2", respectively.

Human prothrombin was isolated from human plasma by barium citrate precipitation and ion exchange chromatography. Each proteolytic fragment treated with Factor Xa was homogenously purified using Mono-Q column chromatography and confirmed by N-terminal amino sequence analysis.

[0023] In Western blot and immunoprecipitation analysis using anti-human prothrombin kringle-2 antibody, human prothrombin kringle-1 did not show any immunological relationship with human prothrombin fragment 2. Also, the anti-angiostatin antibody did not react with any prothrombin fragments.

To evaluate the anti-proliferative effect of purified prothrombin and its derivatives, proteolytic fragments of human prothrombin purified after proteolytic cleavage of prothrombin were assayed for their inhibitory activity on bFGF-stimulated BCE cell proliferation. Prothrombin kringle-1 as well as human prothrombin kringle-2 inhibited bFGF-stimulated endothelial cell proliferation in a dose-dependent manner. Furthermore, after removal of prothrombin kringle-1 or 2, the inhibitory effect on BCE cells can be reversed. However, like angiostatin (a proteolytic fragment of plasminogen containing the kringle 1-4 region), intact prothrombin had no anti-endothelial cell proliferation effect. The angiogenesis inhibitory activity of human prothrombin kringles-1 and 2 was also demonstrated in vivo using the chick chorioallantoic membrane (CAM) assay.

The present inventors have determined that human prothrombin derived from human prothrombin kringle-1
And 2 encoding the E. coli expression vector pET22b ⁺ (Novagen (
Novagen), USA). Expression vectors containing cDNAs encoding prothrombin kringle-1 and prothrombin kringle-2 were called "pEF-hk1" and "pEF-hk2", respectively. The Escherichia coli transformed with pEF-hk2 is referred to as "E. coli BL21 (DE3) / pLysS, phEF13".
M., Yonsei University, Seodaemun-gu, Seoul, Korea, Department of Technology, Department of Food Engineering) and international depositary authorities.

For purification of recombinant prothrombin kringle-2, cultured E. coli BL2
The crude extract of 1 (DE3) / pLysS, phEF13 was chromatographed on DEAE-Sepharose and Sephacryl S-200 columns. It has been demonstrated that recombinant prothrombin kringle-2 suppresses tumor growth in the body.

In order to determine the active region of human prothrombin kringle-2 for inhibiting endothelial cell proliferation, the present inventors synthesized 11 peptides consisting of 15 or 9 amino acids of prothrombin kringle-2. . Prothrombin Kringle-2
A peptide having an appropriate amino acid sequence was synthesized from the N-terminal sequence of the above by overlapping two to five amino acids at the N-terminal and C-terminal of the peptide. As a result, the present inventors have found that the active region of human prothrombin kringle-2 is located between proline 11 and aspartic acid 70.

Hereinafter, the present invention will be further described by the following examples which do not limit the scope of the present invention. Example 1 Purification of Human Prothrombin and Proteolytic Fragments of Prothrombin Complete human prothrombin was purified from human plasma by the improved method of Weinstein et al. (Weinstein, RE et al., Thromb. Res. .,
59: 759-772 (1990)). Sodium citrate was added to a concentration of 0.85% (w / v) to prevent blood clotting and centrifuged at 1,000 xg for 30 minutes to remove residual blood cells and unlysed material. Can be removed. Then, benzamidine-HCl and P-PACK (D-phenylalanyl-L-prolyl-L-ar
Ginine chloromethyl ketone was added to a concentration of 5 mM and 1 M, respectively, and 10 g of QMA Acell (Pharmacia, Sweden) resin was added for ion exchange chromatography. This mixture was incubated at room temperature for 1 hour, and then buffered with 20 mM Tris-HCl buffer (pH 7.5, 100 mM NaCl).
(Including 5 mM benzamidine-HCl and 1 μM P-PACK). The isolated protein was added to a 20 mM Tris-HCl buffer (pH 7.5,
0.6 M NaCl, 5 mM benzamidine-HCl and 1 μM P-PAC
K). The eluted protein is precipitated with barium chloride, and tris buffer (100 mM Tris-HCl, pH 8.0, 150
(containing 1 mM CaCl ₂ mM) and SDS-
PAGE analysis (see: FIG. 1).

For isolation of the prothrombin derivative, 10 mg of purified prothrombin was added at 23 ° C.
Overnight with Factor Xa (Sigma Chemical Co., USA) in a water bath at 4 ° C. in 4 hours against 10 mM Tris-HCl containing 100 mM NaCl and 1 mM PMSF. Dialyzed. This solution was dialyzed once again against 50 mM NaPi, pH 7.0 containing 1 mM PMSF. Each protein fragment was purified to homogeneity using a Mono-Q column pre-equilibrated with 50 mM NaPi, pH 7.0, starting buffer and 0% in 50 mM NaPi.
. Elution was performed with a linear gradient of 1 M NaCl and confirmed by N-terminal amino sequence analysis.

As shown in FIG. 1, after overnight digestion with factor Xa, the four major bands, namely, prothrombin, thrombin, prothrombin kringle-1 (SEQ ID N
O: 1) and prothrombin kringle-2 (SEQ ID NO: 2) by SDS
Observed by gel electrophoresis (see FIG. 1: 1: purified prothrombin, 2: factor Xa
3: Thrombin, 4: Prothrombin kringle-1, 5: Prothrombin kringle-2). Compared to the molecular weight calculated from the amino acid sequence, (1
7 kDa and 13 kDa), prothrombin kringle-1 and 2 were converted to SDS-P
AGEs were run at high molecular weights of 30 kDa and 19 kDa, respectively. This phenomenon is due to rabbit prothrombin kringle-2 and human angiostatin kringle-
Common to several anti-endothelial cell growth molecules, including kringle regions such as
(Lee), TH et al., J. Biol. Chem., 273: 28805-27712 (1998); Cao, Y. et al., J.
Biol. Chem., 271: 29461-29467 (1996)).

Example 2 : Immunological relationship between human prothrombin kringle-1 and 2 Western blot analysis was performed according to the method of Tobin et al. (Towbin, H. et al., Proc. Natl. Acad. Sci. , USA, 76: 4350-4354 (1979))
. The protein was fractionated by 15% SDS-PAGE, and nitrocellulose membrane (Amersham Pharmacia Biotech, Inc.), a nitrocellulose membrane,
US). Blots were blocked for 1 hour with phosphate buffered saline containing 1% bovine serum albumin (BSA) and cultured for 2 hours at room temperature with anti-prothrombin kringle-2 antibody (10 mg / ml) as the primary antibody.
The released primary antibody is washed with a washing buffer (PBS containing 0.5% nonidet P-40).
After washing three times for 10 minutes, each nitrocellulose filter was incubated for 30 minutes with peroxidase-conjugated anti-mouse IgG (Sigma Chemical Co., USA). After washing the released secondary antibody with washing buffer, 4-chloro-
Stained with 1-naphthol solution.

It is already known that anti-rabbit prothrombin kringle-2 antibody does not react with rabbit prothrombin kringle-1 (Lee, TH et al., J. Bi.
ol. Chem., 273: 28805-28812 (1998)). Rabbit prothrombin kringle
Similar to 1, human prothrombin kringle-1 showed no immunoreactivity with human prothrombin kringle-2 by Western blot analysis using an anti-human prothrombin kringle-2 antibody (see FIG. 2: 1: digestion with factor Xa) Prothrombin, 2: thrombin, 3: prothrombin kringle-1 and 4: prothrombin kringle-2). Only intact prothrombin and human prothrombin kringle-2 reacted with the anti-prothrombin kringle-2 antibody.
Moreover, the anti-angiostatin antibody did not react with any of the prothrombin fragments. Immunoprecipitation results were the same as in Western blot analysis,
This means that prothrombin kringles 1 and 2 are not immunologically related and are closely related to the anti-angiogenic effect.

Example 3 Endothelial Cell Proliferation Assay To evaluate the antiproliferative effect of purified prothrombin and its derivatives, proteolytic fragments of human prothrombin were assayed for their inhibitory activity on bFGF-stimulated BCE cell proliferation.

An endothelial cell proliferation assay was performed according to the method of O'Reilly et al. (See O'Reilly, MS et al., Cell, 79: 315-328 (1994)). 10% 10% CO ₂ atmosphere heat treatment inactivated calf serum and 3 ng / ml of recombinant human bFG
Bovine capillary endothelial ("BCE") cells were maintained in DMEM containing F. The cells were cultured in gelatin-coated T75 flasks and cultured at 37 ° C. 0.5
Approximately 12,500 cells per ml were added to each well of a gelatin-coated 24-well plate, and after culturing for 24 hours at 37 ° C, samples were added to each well. After 30 minutes of culture, a final volume of 0.5 m containing 5% calf serum
The medium was added to give 1 DMEM, and bFGF was added to a concentration of 1 ng / ml. After 72 hours of culture, cells were digested with trypsin and the cell count was determined using a hemocytometer. To confirm that the observed inhibitory activity was not due to detachment of cells from the plate, all wells of the assay were examined under an inverted microscope as evidence of cell detachment prior to cell counting.

As shown in FIG. 3, not only human prothrombin kringle-2 but also prothrombin kringle-1 inhibited bFGF-stimulated endothelial cell proliferation in a dose-dependent manner (see FIG. 3). In FIG. 3, (■) shows human prothrombin, (◆) shows thrombin, (x) shows prothrombin kringle-1, and (▲) shows prothrombin kringle-2.

The morphology of BCE cells appeared to resemble regulatory cells or angiostatin-treated cells (Lee, TH et al., J. Biol. Chem., 273: 28805-28812 (1998); (C
ao), Y. et al., J. Biol. Chem., 271: 29461-29467 (1996)). Moreover, the inhibitory effect on BCE cells was mostly reversible after removal of prothrombin kringle-1 or 2.

Example 4 : Chick chorioallantoic membrane assay The chick chorioallantoic membrane ("CAM") assay was used to investigate whether human prothrombin kringles-1 and 2 also inhibit angiogenesis in the body. Poultry fertilized eggs (Puluone, Korea) were cultured at 70% relative humidity with the more gently bent ends up. After culturing at 37 ° C. in a 3% CO ₂ atmosphere for 3 days, a methylcellulose disc containing purified prothrombin kringle-1 or prothrombin kringle-2 (Fischer Scientific,
(USA) was applied to the chick chorioallantoic membrane of each embryo. This disk was prepared by drying 0.45% methylcellulose 10ml in H ₂ O in the presence or absence of prothrombin kringle 1 or 2 on a Teflon (registered trademark) rod. After 48 hours of culture, a 20% intravenous fat emulsion was injected into the chorioallantoic membrane of the chick embryo so that the red color of the blood vessels was clearly contrasted with white. After injection of this fat emulsion, embryos and CAM were observed with a stereoscopic microscope (Nguyen, NM et al., Antic.
ancer Res., 13: 2143-2147 (1993)).

At the dose of 10 mg / disk, both human prothrombin kringles-1 and 2 exhibited a strong inhibitory effect on angiogenesis (see FIG. 4). However, prothrombin and thrombin had little or no effect on BCE cell proliferation. As shown in FIG. 4, Panel A shows PBS treated CA with no signs of reduced angiogenesis.
M. Prothrombin or methylcellulose disc containing thrombin
When mounted on day-old chick germ CAMs, the results were the same as in PBS. However, 10 μg / disk of prothrombin kringle-1 (panel B) and kringle-2 (panel C) reduced the formation of new blood vessels.

Like angiostatin (a proteolytic fragment of plasminogen containing the kringle 1-4 region), the prothrombin fragment as a region of intact prothrombin had no anti-endothelial cell proliferation effect. However, during activation of prothrombin, prothrombin kringles-1 and 2 cleaved with factor Xa exhibited a very strong inhibitory effect.

Example 5 : Comparison of the inhibitory effect of human prothrombin kringle-1 and 2 with other known anti-angiogenic molecules Table 1 shows the relationship between human prothrombin kringle-1 and 2 and other known endothelial cell growth inhibitory molecules. Shows the concentration of 50% inhibition (ED ₅₀ ). Human prothrombin kringle
The ED ₅₀ concentrations of 1 and 2 are approximately 100 nM and 120 nM, respectively. Human prothrombin kringle-1 and 2 are angiostatin (chao (CaO), Y. et al.,
J. Biol. Chem., 271: 29461-29467 (1996)) and rabbit prothrombin fragment 2 (see Lee, TH et al., J. Biol. Chem., 273: 28805-28812 (1998)). Much more potent than other known endothelial cell growth inhibitory molecules, but against bFGF-stimulated BCE cell growth inhibition, plasminogen kringle-5 (chao (C
aO), Y. et al., J. Biol. Chem., 272: 22924-22928 (1997)). However, it has not been reported that the single kringle region of plasminogen and angiostatin, like prothrombin-1 and 2, exists independently in blood. Therefore, human prothrombin kringles-1 and 2 may be the most effective endothelial cell growth inhibitors among naturally occurring single kringle molecules.

[0041]

[Table 1]

Example 6 Expression and Purification of Recombinant Prothrombin Kringles-1 and 2 To clone the cDNA encoding prothrombin kringles-1 and 2, polymerase chain reaction (PCR) was performed with a human prothrombin cDNA library as a template. It was implemented. To perform this PCR, the following single-stranded oligonucleotides were synthesized based on the N-terminal and C-terminal amino acid sequences of human prothrombin kringle-1 and 2, respectively.

Human prothrombin kringle-1: N-terminal primer: 5'-GAATTCCTTCTGGGCCAAGTACACAGC-3 '(SEQ ID NO: 3) C-terminal primer: 5'-AAGCTTTTAGCGTGGAGTCATCGCTAC-3' (SEQ ID NO: 4) Human prothrombin kringle -2: N-terminal primer: 5'-CGAATTCCGAAGGCTCCAGTGTG-3 '(SEQ ID NO: 5) C-terminal primer: 5'-GAAGCTTCTAACGCCCTTCGATGGC-3' (SEQ ID NO: 6)

PCR was performed 30 times at 94 ° C., 57 ° C., and 73 ° C. for 1 minute each, preheated at 72 ° C. for 5 minutes, and finally extended for 10 minutes. The DNA fragment thus obtained was cloned into an E. coli expression vector.

An expression vector (pET22b ′ Novagen, USA) containing a cDNA encoding prothrombin kringle-1 or prothrombin kringle-2 was called “pEF-kH1” or “pEF-hK2”, respectively. The transformed E. coli of pEF-hK2 is called "E. coli BL21 (DE3) / pLysS, phEF13" and is a depositary authority of the Korea Microbial Culture Center (KCCM, 120-749 Seodaemun-gu, Seoul, Korea). (Technical University, Department of Food Engineering) under the deposit number KCCM-10139.

“E. coli BL21 (DE3) / pLysS, phEF13” which is a transformed E. coli of pEF-hK2
Before attacking with IPTG (isopropyl β-D-thiogalactopyranoside)
0.4-1.times. In 600 mM LB medium in the presence of 50 .mu.g per ampicillin.
4O. D. Cultured at unit turbidity. IPTG was added to a final concentration of 0.1-5 mM and the cells were cultured for a further 3 hours. At the end of the incubation period, the bacteria are pelleted by centrifugation at 400 × g for 15 minutes and resuspended in a minimum volume of 10 mM sodium phosphate at pH 7.0 containing 50 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF). I let it. The cells are then sonicated (
(Sonics Material Inc, Danburg, USA). Cell debris is 10 minutes 13
, 500 × g by centrifugation.

To purify the recombinant prothrombin kringle-2, the crude extract was subjected to a DEAE-Sepharose column (2 ×) pre-equilibrated with a buffer containing 10 mM sodium phosphate at pH 7.0 containing 50 mM NaCl. 12 cm). Unbound and weakly bound proteins were removed with 10 mM sodium phosphate buffer containing 180 mM NaCl. After the washing step, proteins were eluted in the buffer at a rate of 20 ml / hr with a NaCl gradient of 0-500 mM. Fractions containing prothrombin kringle-2 were pooled and concentrated on a Centriprep device (Amicon, USA). This concentrated sample was treated with a 50 mM sodium phosphate buffer (pH 7.
Apply to a Sephacryl S-200 column (1.2 × 100 cm) equilibrated in 0).
Elution was carried out at a rate of 8 ml / h.

Amino acid sequence analysis was performed to confirm that the cDNA sequence of the recombinant human prothrombin kringle-2 was faithfully translated.

Example 6 : Inhibition of tumor growth by systemic administration of prothrombin kringle-2 In order for prothrombin kringle-2 to prevent the formation of new blood vessels in the body, the present inventors used primary Lewis implanted subcutaneously. (Lewis) The antitumor effect of prothrombin kringle-2 was investigated for systemically treated C57BL6 mice with lung cancer cells.

[0050] C57BL6 / J male rats (Saniku laboratory: Korea), 6 to 8 weeks old, were used. Rats were habituated into groups of no more than four and their backs were shaved. Six-week-old male rats were stored in partition protection. All rats were provided with animal feed and water ad libitum. The rats were anesthetized with 2 mg ketamine hydrochloride and 0.1 mg xylazine hydrochloride per 20 g mouse. Rats were killed with a lethal dose of methoxyflurane.

[0051] kill the rats that suffered from a tumor of Lewis lung carcinoma of 600~1200mm ^3, was washed a tumor of the skin with betadine and ethanol. Tumor tissue was excised in laminar flow food under sterile conditions. A suspension of tumor cells in 0.9% normal saline was made by passing the live tumor tissue through a sieve and a 26 gauge progressively smaller hypodermic syringe. The final concentration was adjusted to 1 × 10 ⁷ cells / ml and the suspension was placed on ice. After washing with ethanol, the subcutaneous dorsa of the rat
The midline was injected with 1 × 10 ⁶ cells in 0.1 ml saline.

When the size of the tumor reaches 1500 mm ³ , that is, approximately 1 mm after transplantation,
Four days later, rats were arbitrarily extracted into two groups. In one group, the tumor was surgically removed. The incision was sutured with a simple knot suture. The other group underwent an anesthesia and undertook a simulated surgical procedure to manipulate the tumor without damage. Fourteen days later, all rats were dissected and lung surface metastases were counted under a stereomicroscope at 4x magnification.

After resection of the 1500 mm ³ tumor, the abdomen of the rat was treated daily with saline or various concentrations of recombinant prothrombin kringle-2 (0.5 mg / kg and 1 mg / k).
g) was arbitrarily extracted into the subcutaneously injected group, but subcutaneous tumors grew in the midline of the back of each mouse. One group of tumor rats received only the imitation process and was treated with saline injection. After continued treatment for 14 days, all rats were killed and dissected.

As shown in FIG. 5, systematic administration of prothrombin kringle-2 daily resulted in significant suppression of secondary tumor growth within 14 days of treatment (see FIG. 5: 1,
Normal lung; 2, treated with saline; 3, 0.5 mg / kg recombinant prothrombin kringle-2; 4, 1 mg / kg recombinant prothrombin kringle-2).
FIG. 6 is a graph showing weight loss of lungs treated with recombinant prothrombin kringle-2.

Fourteen days after the treatment, rats treated with 1 mg / kg of prothrombin kringle-2 were found to have an average of 90% or more suppression of primary tumor growth (n = 10). In contrast, tumors grew rapidly in rats treated with saline during the same 14-day treatment period (n = 8).

Example 7 : Anti-Endothelial Cell Proliferation Activity of Prothrombin Kringle-2 Peptide In order to determine the endothelial cell growth inhibitory activity of human prothrombin kringle-2, 11 peptides consisting of 15 or 9 amino acids of prothrombin kringle-2 were used It was synthesized by Peptron Corp. (Korea). From the N-terminal sequence of prothrombin kringle-2, a peptide having an appropriate amino acid sequence was
It was synthesized by overlapping the terminal and C-terminal 5 amino acids (see Table 2).
On the other hand, due to problems in the synthesis and superposition of the diamino acids, SEQ ID NO: 1
The second peptide is synthesized so that it can contain only 9 amino acids. The synthesized peptide was purified by high performance liquid chromatography (HPLC).

[0057]

[Table 2]

An endothelial cell proliferation assay was performed according to the method of Example 3. As shown in Table 2, the peptides of SEQ ID NOs: 8 to 13 suppress bFGF-stimulated endothelial cell proliferation, and the inhibitory activity of SEQ ID NO: 12 is most effective. Therefore, SEQ ID NO. It was clearly determined that 12 peptides played important roles in exhibiting anti-endothelial cell proliferation effect.

Administration and Effective Dose The effective dose of human prothrombin kringle-1,2 or a functionally equivalent peptide will depend on the age, weight and progression of the disease of the patient, but will be less than 0 per dose.
. It is preferred to administer parenterally at 8-2.0 mg / kg / day, but this can be individualized by the experience of those skilled in the art.

[0060]Acute toxicity Of human prothrombin kringle-1, 2 or a peptide functionally equivalent thereto
To test for acute toxicity, C57BL / 6J male rats were treated with human prothrombin
Subcutaneous injection of Angular-1, 2 or peptide, counting dead ones and LD for 7 days ₅₀ It was determined. As a result, LD₅₀Was determined to be approximately 5 g / kg, which was
Antibodies comprising toprothrombin kringle-1,2 and functionally equivalent peptides thereof
This shows that the tumor agent is sufficiently safe within the effective dose range.

The pharmaceutical composition of the present invention comprising the active ingredient of human prothrombin kringle-1, 2 or a peptide functionally equivalent thereto and a pharmaceutically acceptable carrier can be administered by injection. The injection form can further comprise isotonic aqueous solutions or suspensions and pharmaceutically acceptable carrier covers preservatives, stabilizers, wetting agents, emulsifiers, salts or buffers that change the osmotic pressure.

Although the present invention has been illustrated and described with reference to a specific embodiment using Lewis lung cancer as a test tumor, the antitumor agent of the present invention can be used for skin cancer, laryngeal cancer, uterine cancer, colon cancer, lung cancer and bone marrow. It is also effective for various tumors such as cancer.

As apparently exemplified and proved above, the present invention relates to human prothrombin kringle-1, 2 having an amino acid sequence of human prothrombin kringle-2 having an anti-endothelial cell proliferation effect, and peptides functionally equivalent thereto A cDNA encoding human prothrombin kringles-1 and 2 derived from human prothrombin, a recombinant expression vector containing the cDNA, and a human prothrombin kringle-2
A recombinant microorganism transformed with a recombinant expression vector representing human prothrombin kringles -1 and 2 from human plasma, and a method for producing recombinant human prothrombin kringle -2 from a recombinant microorganism; and Provide pharmaceutical applications of the protein. The anti-endothelial cell growth molecule, human prothrombin kringle-1, 2 and functionally equivalent peptides, should be used for cancer treatment, since anti-vascular therapy on endothelial cells of the tumor should induce little or no drug resistance. Is the most reliable candidate.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a photograph showing a human prothrombin derivative analyzed and purified from human plasma by SDS-PAGE.

FIG. 2 is a photograph showing Western blot analysis of human prothrombin and its derivatives.

FIG. 3 is a graph showing the endothelial cell growth inhibitory effect of human prothrombin and its derivatives.

FIG. 4 shows the anti-angiogenic effect of human prothrombin kringle-1 and 2 on CAM.

FIG. 5 is a photograph showing suppression of tumor growth of human prothrombin kringle-2.

FIG. 6 is a graph showing suppression of tumor growth of human prothrombin kringle-2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 (C12P 21/02 C C12R 1:19) // (C12P 21/02 C12N 15/00 ZNAA C12R 1:19) A61K 37/475 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Kim Sung Soo South Korea, Seoul 159 −050, Yancheong, Mokdong, Mokdong Apartment 313 -1103 (72) Inventor Im Tae-yong South Korea, Seoul 137-070, Yongsank, Icheon-dong 407, Hungarian Part 214-1906 (72) Inventor Park Chang-South Korea, Seoul 135-921, Kannan H, Yeoksandon 728-48, 301 CB06 CC24 CE07 CE11 DA01 4B065 AA26X AA93Y AB01 AC14 BA02 CA24 CA44 4C084 AA02 AA07 BA44 CA18 DC12 NA14 ZB26 4H045 AA10 AA20 AA30 BA13 BA14 BA15 BA16 BA17 CA42 DA65 EA28 FA72 FA73 FA74 GA22 GA23

Claims (14)

[Claims] 1. The amino acid sequence of human prothrombin kringle-1 represented as SEQ ID NO: 1. 2. A cDNA encoding the human prothrombin kringle-1 of claim 1 (SEQ ID NO: 18). 3. The amino acid sequence of human prothrombin kringle-2, represented as SEQ ID NO: 2. 4. A cDNA encoding the human prothrombin kringle-2 of claim 3 (SEQ ID NO: 19). 5. A cDNA expressing human prothrombin kringle-1 (S
A recombinant expression vector pEF-hK1 comprising EQ ID NO: 18). 6. A cDNA expressing human prothrombin kringle-2 (S
A recombinant expression vector pEF-hK2 comprising EQ ID NO: 19). 7. Escherichia coli BL21 (DE3) / pLysS, phEF13 (KCCM-10) transformed with the recombinant expression vector pEF-hK2 of claim 6.
139). 8. A peptide derived from human prothrombin kringle-2, which inhibits proliferation of endothelial cells. 9. The human prothrombin kringle-2 having an appropriate sequence of 5 to 20 amino acids from proline 11 to aspartic acid 70 of human prothrombin kringle-2.
Peptide. 10. A method for isolating and purifying human prothrombin from human plasma, and (ii) treating human prothrombin with factor Xa to obtain prothrombin kringle-protein.
A method for producing prothrombin kringles-1 and 2 from human plasma, comprising the steps of providing 1 and 2; and (iii) isolating prothrombin kringles-1 and 2 by ion exchange chromatography. 11. A method comprising the steps of: (i) culturing a recombinant microorganism transformed with the expression vector containing the cDNA of claim 4, and (ii) applying ion exchange and gel filtration chromatography to the culture. Obtaining a recombinant human prothrombin kringle-2. 12. The recombinant microorganism is Escherichia coli BL21 (DE3) / pLy.
The production method according to claim 11, which is sS, phEF13 (KCCM-10139). 13. An antitumor agent comprising an active ingredient of human prothrombin kringle-1 or 2, or a peptide functionally equivalent thereto, and a pharmaceutically acceptable carrier. 14. The antitumor agent according to claim 13, which is effective for skin cancer, laryngeal cancer, uterine cancer, colon cancer, lung cancer and bone marrow cancer.